Nucleolar organiser region (NOR) location in karyotypes of Australian ground frogs (Family Myobatrachidae)

M. J. Mahony & E. S. Robinson School of Biological Sciences, Macquarie University, North Ryde, New South Wales 2113, Australia

Abstract

Nucleolar organiser regions (NORs) were examined in over 90~ of the species of Australian ground frogs (familiy Myobatrachidae), including representatives from all twenty currently recognised genera and the three subfamilies. Throughout the family, location of the NOR within the karyotype showed considerable variation yet karyotype morphology showed uniformity. The precise mechanism(s) whereby variation in NOR location evolved while karyotype morphology was unchanged remains uncertain. Comparison of the two major sub- families showed that the Limnodynastinae had a greater diversity of NOR location than the Myobatrachinae. The limnodynastine genus, Heleioporus, was the only one to show multiple NOR sites in several species. NOR location was particularly stable within most polytypic genera. Differences in NOR location within the remaining polytypic genera (Heleioporus, Limnodynastes, Neobatrachus, Philoria'and Ranidella) pointed to taxonomic discriminations that were generally consistent with recent proposals based on other criteria.

Introduction

Ribosomal DNA genes, usually present as tan- dem repeats, code for the rRNA of interphase nuleoli and the region of a chromosome containing these genes is termed the nucleolar organiser region (NOR). NORs can be easily detected in metaphase chromosomes by a quick and simple treatment with silver nitrate (Goodpasture & Bloom, 1976) which appears to stain nucleolar phospho-proteins B23 and C23 associated with rDNA transcription (Schwarzacher & Wachtler, 1983, Busch et al., 1982). A few examples of silver staining of chro- mosomal regions other than NORs have been reported (Varley & Morgan, 1978; Medina et al., 1983; Haaf et al., 1984) but in the vast majority of cases it is specific for those rDNA genes that were actively transcribing during the previous inter- phase. A comparison of silver stained mitotic metaphase spreads with those conventionally stained with for example, aceto-orcein or Giemsa

shows that NORs are usually located within regions long referred to as secondary constrictions. Silver stained NORs are however more readily identified tllan are secondary constrictions and not all secon- dary constrictions silver stain (see later). It is the specificity of silver staining for a complex gene lo- cus that makes the NOR potentially useful as a chromosome marker for cytogenetic and cytotaxo- nomic studies.

In studies which have investigated the location of the NOR in anurans one feature has consistently emerged; in closely related species (species com- plexes or species groups), the NOR is almost always localised in the same region of the same chromo- some pair. Schmid (1983) has argued that, 'excep- tions to this rule gave indications of chromosomal rearrangements having occurred in the NOR- carrying chromosome segments in the evolution of the Anura'. Schmid (1978 a & b, 1982) examined species from a wide variety of genera and families, while King (1982) concentrated on the members of

Genetica 68, 119-127 (1986). ~ Dr W. Junk Publishers, Dordrecht. Printed in The Netherlands.

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a single but large and diverse genus. To date no study has examined NOR location in an extensive array of genera from one family. The Australian myobatrachid (ground) frogs are a suitable group for such a comparative study of NOR location. These frogs are generally recognised as an old southern fauna which has evolved in isolation on the Australian continent since the breakup of Gondwanaland. Furthermore, this family has in re- cent times been the subject of comparative studies in morphology, biochemistry and behavior, which have resulted in various taxonomic revisions. Karyotypic analysis has provided little assistance in taxonomic resolution because of the well known stability in diploid number, relative chromosome lengths and morphology. However, Morescalchi and Ingram (1978) used the limited karyotypic data to point to some generic affinities. For a summary of the limited previous work on the chromosomes of myobatrachid frogs see Mahony and Robinson (1980).

The present study was undertaken (a) to establish the extent of variation in NOR location in a large number of genera and species of Myobatrachidae, and (b) to determine whether NOR location has any taxonomic and phylogenetic value, particularly at the generic level, in a family where the current state of nomenclature and phylogenetic relation- ships are distinctly unstable (Tyler, 1979).

Materials and methods

Animals

Frogs were collected between 1979 and 1984 from localities across the Australian continent with most sites in the south-eastern and south-western regions. Identifications were confirmed where necessary from Cogger (1983) and the taxonomy adopted here is the same as that of Cogger et aL, (1983). Identified specimens have been deposited in the Australian Museum, Sydney, New South Wales.

The chromosomes of the following species were examined.

Subfamily Limnodynastinae Adelotus Ogilby, 1907; A. brevis, Heleioporus

Gray, 1941; H. albopunctatus, H. australiacus, H. barycragus, H. eyrei, H. inornatus, H. psammophi-

lus, Lechriodus Loveridge, 1935; L. fletcheri, L. aganoposis, L. melanopyga, Limnodynastes Fit- zinger, 1843. (peroni group)L, convexiusculus, L. fletcheri, L. peroni, L. tasmaniensis, (dorsalis group) L. dorsalis, L. dumerillii, L. interioris, L. terraereginae, (ornatus group)L, ornatus, L. spen- ceri," L. salmini. Megistolotis Tyler, Martin & Da- vies, 1979; M. lignarius. Mixophyes Gunther, 1864; M. balbus, M. fasciolatus, M. iteratus, M. schevilli. Neobatrachus Peters, 1863; N. aquilonius, N. cen- tralis, N. pelobatoides, N. pictus, N. sudelli, N. su- tor, N. wilsmorei. Notaden Gunter, 1873: N. benet- tii, N. melanoscaphus, N. nichollsi. Philoria Spencer, 1901; P. frosti, P. kundagungan, P. Ioveridgei, P. spagnicolus.

Subfamily Myobatrachinae Arenophryne Tyler, 1976; A. rotunda. Assa Tyler,

1972; A. darlingtoni. Crinia Tschudi, 1938; C. geor- giana, Geocrinia Blake, 1973; G. leai, G. laevis, G. lutea, G. rosea, G. victoriana. Metacrinia Parker, 1940; M. nichollsi. Myobatrachus Schlegel, 1850; M. gould#. Paracrinia Heyer and Liem, 1976; P. haswelli. Pseudophryne Fitzinger, 1843; P. austra- lis, P. bibroni, P. coriacea, P. corroboree, P. dendyi, P. guentheri, P. major, P. occidentalis, P. semimar- morata. Ranidella Girard, 1853; R. bilingua, R. glauerti, R. insignifera, R. parinsignifera, R. pseudinsignifera, R. remota, R. riparia, R. sig- nifera, R. tasmaniensis. Taudactylus Straughan & Lee, 1966; T. acutirostris, 77. eungellensis, 77. liemi, T. rheophilus. Uperoleia Gray, 1841; U. crassa, U. inundata, U. laevigata, U. lithomoda, U. rugosa.

Subfamily Rheobatrachinae Rheobatrachus Liem, 1973; R. vitellinus

Techniques

Live sl~ecimens were transported to the laborato- ry and processed as soon as possible after capture. Mitotic spreads were obtained from duodenal epithelium using a technique described by Mahony and Robinson (1980). At least five karyotypes were prepared and measured for each species usually from several specimens. Relative lengths of chro- mosomes were calculated as a percentage of total haploid length. For silver staining of mitotic spreads the method of Bloom and Goodpasture (1976) was used.

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Results

L L i m n o d y n a s t i n a e

C h r o m o s o m e n u m b e r is not un i fo rm t h r o u g h o u t this subfamily . Mos t o f the 40 species examined have a d ip lo id n u m b e r o f 2n = 24 but a few have 22. Karyo types o f the m a j o r i t y o f genera show two dis- t inct size classes: c h r o m o s o m e s 1 to 6 are large

(Relat ive Length range 16% to 80/0) while the re- m a i n d e r are smal ler (R.L. range 7% to 4%). Mos t c h r o m o s o m e s are metacen t r i c but submetacen t r i cs and subacrocen t r i cs are qui te c o m m o n and acrocent r ics occur in a few species. M i c r o c h r o m o - somes and he t e romorph i c sex c h r o m o s o m e s were not observed. A typica l ka ryo type ( 2 n = 2 4 ) is shown in Fig. 1.

In Table 1 the site o f the N O R is represented di-

a

I I ;; ;; xx 6 !

b

1 4 6

7 12

C

I

X | X X * 8 * * *~* * * 7 12

r "1

Fig. 1. Typical karyotypes of the three subfamilies: (a) Limnodynastinae (Adelotus brevis);-(b) Myobatrachinae (Pseudophryne cor- roboree);-(c) Rheobatrachinae (Rheobatrachus vitellinus). The NOR bearing pair is shown to the right in each case. Arrowheads indi- cate the major secondary constrictions and arrows the silver stained NORs. Scale bar represents 10 u.m.

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Table 1. Variation in NOR location in the subfamily Limnodynastinae (excluding the genus Heleioporus).

Genus Number of species Haploid chromosome number and NOR location

Total Examined 1 2 3 4 5 6 7 8 9 10 11 12

A delotus 1 1 II

Lechriodus 4 3

Limnodynastes peroni group 5 4

dorsalis group 5 5 LI

ornatus group 2 2

(L. salmint) 1

Megistolotis 1 1

Mixophyes 4 4

Neobatrachus 7 6

iN. centralis) 1

Notaden 3 3

Philoria 4 3

II

(P. frostO 1

1 2 3 4

1 2

o

(-)

(-)

(-)

5 6 7 8 9 10 11 12

5 7 9 10 11 12

agrammatically for each genus (except Heleioporus which is shown in Table 2). Location of the NOR is quite variable, with 13 different sites and 8 chro- mosomes involved (Table 1). No particular pair or chromosomal site appears to be favoured.

The two monotypic genera (Adelotus, and Megistolotis) have distinctive NORs as do the poly- typic genera Lechriodus, Notaden and Mixophyes. NOR location is variable within four of the poly- typic genera: in both Neobatrachus and Philoria a single species is different; in Limnodynastes recog- nisable species groups are involved; and in Heleioporus variation includes multiple NOR sites in addition to differences in NOR location.

In Table 2 the site of the NORs is represented di- agrammatical ly for each species of Heleioporus. Four species, H. albopunctatus, H. eyrei, H. inora- tus and)H, psammophilus, have NORs in the same position on chromosomes 1, 2, 3 and 11. H. al- bopunctatus and H. eyrei have an additional NOR on chromosome 4. H. australiacus has only one NOR site, but it is notable that this site is the same as that observed on chromosome 11 in the four spe- cies above. The remaining species, H. barycragus, has only one NOR, which is not located in any of the sites identified in the other species.

Table 2. NOR location and chromosome morphology within the genus Heleioporus.

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Species Ch romosome morphology and NOR location

1 2 3 4 5 6 7 8 9 10 11 12

H. albopunctatus

H. eyrei

H. inornatus

H. psammophilus

H. australiacus

H. barycragus

H~H~HHHHHH~

H~NHHHH~~ HHNHHHHH~,~ HNHHHHH~~

a

1 2 3 6

7 11 12

r !

b

-11-1t-Ii it I I ! 2 3 6

I | i I I I s~ .,Jr ~. 7 11 12

r I

Fig. 2. Karyotypes of Heleioporus psarnmophilus stained with (a) aceto orcein, (b) silver. Arrowheads indicate the major secondary constrictions and arrows the silver stained NORs. Scale bar represents 10 p.m.

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IL Myobatrachinae

All 39 species examined had a diploid number of 2n = 24 and little variation in karyotype morpholo- gy was observed throughout the subfamily. Almost all species possessed a karyotype similar to that shown (Fig. 2), with two size classes: a group of larger chromosomes - Nos. 1-6 (Relative Lengths range 15~ to 9~ and a group of smaller chromo- somes - Nos. 7-12 (R.L. range 7~ to 4~ The only clear departure from this standard karyotype was that in a few genera chromosome 12 was dis- tinctly smaller than the rest.

In Table 3 the site of the NOR is represented di-

agrammatical ly for each genus. Location of the NOR is more stable than that observed in the Lim- nodynast inae with 6 of the 12 genera having the NOR on the short arm of chromosome 4, but the variation is still considerable with the NOR located on 5 chromosomes in the remaining genera.

Five genera are polytypic; Geocrinia, Pseu- dophryne, Taudactylus, Uperoleia and Ranidella and with the exception of Ranidella, NOR location is stable within each genus. In Ranidella, two spe- cies, namely R. remota and R. tasmaniensis have NOR locations different from one another and from other members of the genus.

Table 3. Variation in NOR location in the subfamily Myobatrachinae.

Genus Number of species

Total Examined

Haploid chromosome number and NOR location

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

A renophryne I 1

A ssa 1 1

Crinia 1 1

Geocrinia 5 5

Metacr in ia 1 1

M y o b a t r a c h u s 1 1

Paracrinia 1 1

P s e u d o p h r y n e 10 10

Ranidel la 13 7

(R. rernota) 1

(R. tasrnaniensis) 1

Taudac ty lus 5 4

Uperoleia 15 6

H

H H

H M

2 3 4 5 6 7 8 9 1 0 1 1 1 2

2 4 5 1112

III. Rheobatrachinae

This subfamily includes only the genus Rheo- batrachus which until recently consisted only of the type R. silus. The karyomorphology of R. silus was presented by Morescalchi and Ingrain (1974). A new species, Rheobatrachus vitellinus, found and identified by the senior author has been described and the chromosomes of one specimen have been examined (Mahony et al., 1984).

Both Rheobatrachus species have a diploid num- ber of 2n=24, with the chromosomes in two size classes: a group of larger chromosomes - Nos. 1 to 6 (Relative Length range 15% to 9%) and a group of smaller chromosomes - Nos. 7 to 12 (R.L. range 6% to 3%). Variation in chromosome morphology occurs between R. silus and the newly described species; chromosome 6 is acrocentric, and 10 meta- centric in R. silus (Morescalchi & Ingram, 1974), whereas in the newly described species they are sub- metacentric and acrocentric respectively. The NOR is located on the short arm of pair 6 in the new spe- cies, but its position in R. silus is unknown.

Discussion

Viewing the family as a whole, the major conclu- sion to be drawn from this comprehensive survey of myobatrachid karyotypes is that diversity in NOR location is accompanied by uniformity in karyo- type morphology. No particular chromosome, or chromosome arm appears to show a preference for NOR location, indeed the NOR is found on almost all members of the haploid complement but rela- tive chromosome sizes and centromere positions were remarkably constant. To account for this NOR variation throughout the family against a background of karyotypic homogeneity, the 'con- ventional' types of restructuring such as transloca- tions and inversions are unappealing mechanisms, since they would be expected to lead to a spectrum of changes in karyotype morphology. Other mechanisms, for example multiple cryptic structur- al rearrangements or minute insertions (King, 1980), reintegration of amplified rDNA genes dur- ing oogenesis (Nardi et al., 1977; Schmid, 1978) or activation of latent nucleolar sites (King, 1980) may well be involved in myobatrachids but in the ab-

125

sence of adequate G-banding data the actual source of NOR transposition or activation remains uncer- tain. Unfortunately these animals have proved to be extremely resistant to adequate G-banding.

Early in this study it became clear that NOR lo- cation was uniform within many lower-order taxa. Schmid (1978 a, b) and King (1980) had already ob- served in other anuran families that NOR position was stable amongst closely related species or within species complexes. As our work on myobatrachids expanded, we found that NOR uniformity extended to the generic level. Indeed, so consistent was NOR location within almost all polytypic genera that we examined the status of exceptional species in detail to see if NOR position could be a useful taxonomic indicator. We consider that the NOR evidence sup- ports some generic groupings not widely accepted in the current literature.

Perhaps the clearest example of NOR uniformity reflecting generic limits is shown in the genus Lim- nodynastes, the type genus of the subfamily Lim- nodynastinae. This genus is currently divided into three species groups, namely the 'peronf, 'dorsalis' and 'ornatus' groups although Tyler et al., 1979 suggested that the groups 'possibly merit elevation to generic status' based on internal morphology. The NOR data support this elevation in that the members of each group share an NOR location dis- tinct from the other two groups. The degree of difference in NOR position between the three groups is at least as great as differences between many genera in the family and in the case of the 'dorsalis' and 'ornatus' group, generic status is fur- ther strengthened by a difference in chromosome number. Names are available for the three groups (proposed earlier on morphological grounds) and the following suggestions are made: Lim- nodynastes Fitzinger, (sensu stricto) 1841 refers to the 'peronf group; Platyplectron Peters, 1863 is the first available name to refer to the 'dorsalis' group; and Platyplectrum Gunther, 1863 refers to the 'or- natus' group. One species currently regarded as a member of the 'peronl ~ group (Limnodynastes salmint) has a sufficiently distinct NOR location to warrant inclusion in a new genus.

Several other generic revisions in the subfamily Limnodynastinae are favoured on the basis of NOR location. A division of Philoria agrees with the recognition of a separate genus Kyrannus Moore, 1958 for the three species that have their

126

NOR on chromosome 1, but the validity of the ge- nus name is under question (Cogger, 1983). A divi- sion of Neobatrachus would resuIt in generic sepa- ration of N. centralis and in the genus Heleioporus, one species, H. barycragus shows a distinctive site and may also warrant separate generic status.

In the subfamily Myobatrachinae NOR location is particularly stable within genera with the excep- tion of two species of Ranidella (R. remota and R. tasmaniensis). The genus Raniclella has been the centre of a number of morphological studies and the generic affiliations of several species remain un- certain (see Thompson, 1981). In fact, even the use of the name Ranidella (rather than Crinia) has re- cently been challenged (Heyer et al., 1983). On the basis of NOR location, R. remota and R. tas- maniensis should be removed from the genus Ranidella.

Proposals regarding the phylogenetic relation- ships of myobatrachid genera based on NOR loca- tion alone cannot be made without a knowledge of the chromosomal mechanisms involved and their temporal sequence. However, some general state- ments can be made about likely ancestral and der- ived states, particularly when taken together with some recent microcomplement fixation (MCF) data of Daugherty and Maxson (1983). For example, the genera in the subfamily Myobatrachinae are more conservative in NOR location than the Lim- nodynastinae. Six myobatrachine genera have the NOR located on the same arm of the same chromo- some and in four of these genera (Arenophryne, Metacrinia, Myobatrachus and Pseudophryne) it appears to be in exactly the same position. Im- munological studies (Daugherty & Maxson, 1983) indicate that these genera are closely affiliated and that the primary generic diversification within the Myobatrachinae may have occurred as long ago as the Cretaceous, thus predating the diversification of the extant mammalian orders.

In sharp contrast, the subfamily Limnodynasti- nae is notably diversified in NOR location and in- cludes the only example of a genus with multiple NOR sites (Heleioporus). Multiple sites are not un- common in mammals (Goodpasture & Bloom, 1975) but in over 90O7o of frogs examined, the NOR is found to occur at only one locus (Schmid, 1982a & b). The origin and functional significance of ad- ditional NOR sites is still a matter of conjecture. In this case, as in others (Hsu et al., 1975), it would

appear that they represent a derived condition. The evidence in Heleioporus indicates that their origin in four of the species, H. albopunctatus, 14. eyrei, H. inornatus and H. psammophilus, occurred in their ancestor and has been maintained subsequent to their speciation. Taken together with examples of reduced diploid chromosome number and of poly- ploidy, the Limnodynastinae, while still showing conservatism in chromosome morphology, are much less conservative karyotipically than the My- obatracbinae.

Acknowledgements

We gratefully acknowledge the help provided by numerous people with the collection of specimens, especially M. Davies, S. Donnellan, H. Ehrmann, D. Roberts and M. Tyler. Several useful taxonomic suggestions were made by H. Cogger.

We also wish to thank E G. Johnston for reading the manuscript, R. Oldfield for advice with pho- tography and M. Minard for secretarial help.

Support for this study was provided by the Aus- tralian Biological Resources Study, Australian Museum Postgraduate Research Awards and the Peter Rankin Trust Fund.

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Received 10.1.1985. Accepted 13.6.1985.